Process for preparing toyocamycin



United States Patent Ofi" 3,115,222 Patented Dec. 31, 1963 ICC 3 116 222 Princess FOR rauiAnINc TUYGCAMYCIN Koppaka V. Rao, Pine Brook, William S. Marsh, Wanaque, and Donald W. Benn, River Vale, N.J., assignors 2 ganism, regardless of its appearance or physiological behavior, that may be developed by means of transformation, transduction, genetic recombination or some other genetical procedure or equivalent material, whereby it has acquired the ability to produce the elaboration prod- Gelatin skimmed Milk Lacking Moderate; whitish.

to Chas. Pfizer & Co., Inc New York, N.Y., a corpo- 5 ration of Delaware uct here described or to carry on the biochemical change No Drawing. Filedocts,19525enNm228745 here described. For the preparation of toyocamycin, 7 Cl i ((31, 195 .30) a wide variety of fermentation media are satisfactory. A medium composed basically of a source of nitrogen, This invention relates to the cultivation under con- 10 a carbohydrate and minerals is required. Satisfactory trol conditions of a strain of Streptomyces rimosus, to nitrogen sources include hydrolyzed casein of various the biologically active substance produced thereby, the types, soy bean meal, distillers solubles, corn meal, nianti'oiotic toyocamycin, and to the methods for its retrates, ammonium salts, urea and the like. Suitable covery and concentration from crude solutions includcarbohydrate sources are corn starch, dextrose, lactose, ing fermentation broths. dextrin, etc. The preceding materials frequently contain This invention relates specifically to the preparation suflicient minerals to satisfy the mineral requirement of and isolation of toyocamycin from Streptomyces rithe organism without substantial addition of mineral mosus. Toyocamycin possesses known antimicrobial components. A suitable aqueous nutrient medium conactivity for a variety of organisms which was described tains 10 g./l. dextrose monohydrate, 15 g./l. dibasic poby Nishimura et al. in l. Antibiotics, Ser. A9 (2), 60-62 tassium phosphate, 2 g./l. sodium chloride, 2.5 g./l. dis- (l956). In addition to this utility, toyocamycin is also tillers solubles and 2 g./l. of calcium carbonate. The known to be elfective in inhibiting the growth of maligpl-l of the fermentation is most suitably maintained henant tumors. tween 6.5 and 7.5. After autoclaving and inoculating,

The organism which is employed in the valuable procthe fermentation is carried out at a temperature usualess of the present invention was isolated from a soil 1y between 26 and about 36 C. with aeration. The sample on an agar formulation of the following compoprogress of fermentation is followed by testing the cytosition: toxic activity of the samples by the Toplin method, as G./l. described in Cancer Research, 19, 959 (1959), or by Dextrose 2D the Eagle-Foley growth method as described in Cancer Asparagine 0,2 30 Resend}, 1017 c Beef exiract 0.25 For the commercial production of antibiotic by this Dipotassium acid phosphate 05 process, submerged culture in tne usual type of equip- Agar 15 ment similar to those in the art 18 employed. Suitable tanks varying in size from 2000 gallons to 20,000 gal- The isolated culture was examined and found to be has or m equipped with emcient agitation and means a typical Streptomyces and was identified by DL B. for aseptic aeration of the contents with up to two or Routien as a member of the species Streptomyces rimore voiumfis of alr P mmute are m gr A satis' mosbm This culture was keyed using the key factory medium for large scale production 18 given above. provided by Waksman in his book, The Actinomycetes, Thehrowth of the micfoofganlsm and antibiotic P vol. 2, 1961. A culture of this strain has been deposited 40 ducilOn usually rfiachfis Its maxlmum after about to with the American Type Culture Collection of Wash- 72 hours at about to about However Vari ington, DC and given the ATCC 14500 ation in equipment used, the rate of aeration, stirring,

The culture characteristics of the culture are set forth e c. frequently affect the time period for maximum acin Table I. The results are based upon six tubes or tivity to be reached. A period of at least 24 hours is plates of each medium incubated for two weeks. 45 required in any case. Aeration of the medium on sub- TABLE I Medium Growth Aerial Mycelium and Soluble Remarks Sporulation Pigment Glucose-asparagine agar Good Lackin Lacking..- Vcgctativcmycelium pale yellow to cream- Synthctic agar Good,i1at. Scent, too little to do.. Vcgct i i c mycelium colorless. Chains of show color. spores in loose spirals and sometimes Nutrient agar- Good T nkin do Vdg efa iive myceliurn cream-yellow. Glucose agar d0 Poor; white do Vegetative myceliurn cream-yellow; very Pr hams yeast extract agar -do Moderate; white do vgli iiim mycclium yellow; vcry rimose.

Slight liquefaction. Coagulation and peptcnization. pH

changed from 6.4 to 7.0.

Potato Plug Good; whitish gray--- -do Vegetative mycelium yellow-brown to yelow' rnnose. Starch Plate Fair Good; white do No hydrolysis.

N 0 growth Nitrlzgtcs reduced to nitrites.

0. Calcium Malatc Fair Lacking Lacking Vegetative mycelium yellow. Slight digestion ol calcium malate.

Peptone Iron Agar- It is to be understood that for the production of the antibiotic toyocamycin, according to the present invention, it is also intended to include mutants produced from this organism by various means such as radiation with X-ray, ultra-violet light, treatment with nitrogen mustards and the like. We wish also to include any or- N 0 HZS produced.

merged growth is maintained at the rate of about /2 to 2 volumes of air per volume of broth per minute. Aseptic conditions, of course, must be maintained throughout the transfer of the inoculum and the growth of the microorganism.

After a satisfactory level of the antibiotic is attained,

the active substance may be isolated by procedures well known to those skilled in the art. For example, the antibiotic activity may be extracted from the filtrate broth at a pH from about 4 to about 6 with an alkanol. The solvent extract may then be concentrated to remove most of the alkanol. This concentrate is freeze dried and the solid extracted with an alkanol. The extract is passed through a chromatographic column for example a chromatographic column containing a weakly basic anion exchange resin. Thereafter, the column is washed with an allcanol. In this manner, the active substance goes through the column while most of the dark colored impurities remain absorbed thereon. The effluent fractions containing the active material are then combined and concentrated to a small volume causing the bulk of the active antibiotic to precipitate as a solid crystalline product. The active component toyocamycin may be further purified by recrystallization, for example, from a 1:1 mixture of methanol and chloroform.

Another method which may be employed to isolate the active substance is to stir the filtered broth from about 0.5 to about one hour with about 2% of activated carbon. The activated carbon is removed by filtration and the cake is washed and eluated twice with a mixture containing three parts of an alkanol and two parts of an equal mixture of water and pyridine. Thereafter, the combined cluate is concentrated to remove most of the organic solvents and is passed through a chromatographic column. Florisil an activated magnesium silicate which is prepared according to the procedure described in U.S. Patent 2,393,625 has been found to be of particular value when used as the chromatographic material. The column is then washed with water and the active material is eluted with an alkanol. The combined eluate is concentrated causing the crystallization of the active component. Toyocamycin may be further purified by recrystallization by the procedure described above. The term alkanol as employed in the aforementioned procedures consists of those alcohols containing from one to six carbon atoms.

The antibiotic of this process is obtained as a colorless crystalline solid which has a melting point of 247 250 C. The elemental analysis is: 49.14% carbon, 4.47% hydrogen and 24.00% nitrogen, and it demonstrates a characteristic ultraviolet spectral with maxima at 232 and 278 m with values of 355 and 535 respectively. The infrared spectrum was measured in a potassium bromide pellet containing 1.0% of the antibiotic; the most characteristic band in the infrared spectrum is at 45 This antibiotic is sparingly soluble in water, methanol, ethanol, isopropyl alcohol, butanol, acteone and ethyl acetate. It is soluble in dilute acids and alkali and gives a negative ferric chloride and a faintly positive ninhydrin reaction.

The chemical and physical properties described above agree with those given for toyocamycin, as described in the Journal of Antibiotics at 8A, 145 (1955) and 8A, 189 (1955).

Toyocamycin produced by the process of this invention Was tested and found to be effective in treating a number of different types of malignancies. It was tested for anti-leukemia activity against a transplantable lymphoid leukemias, known as L-1210, in Swiss white mice weighing from 18 to 22 grams, according to the procedure described by LaW, L. W., inthe Journal of Natl. Cancer Inst, vol. 10, page 179 (1949). Ll210 was injected intraperitoneally with ascitic fluid diluted (after a cell count) to a concentration of 10 cells/ml. Each animal received 0.1 ml. of [diluted fluid (10 cells) and the aninals were divided into groups of six. Intraperitoneal adminstration of toyocamyc-in, dissolved in 0.85% aqueous saline, in various doses at various times was began 24 hours after the injection of the L12-10. One group of animals which had been injected with L12l0 was maintained as a control and received injections containing only 0.85% saline. The study was continued until all the animals died. During this time, the change in body Weight was recorded and, thereafter, a comparison of the survival time was made of the animals treated with toyocamycin and the untreated animals.

The activity of toyocamycin is particularly pronounced against human carcinoma cells (strain HeLa) grown in a tissue culture according to the procedure described by Rightsel et al. (Journal of Immunology, vol. 76, pages 464-474, 1956). In this test, the cytotoxic activity of toyocamycin is detected at levels as low as 0.018 mcg./ ml. This compares well with other highly cytotoxic compounds such as actinomycin D at 0.10 meg/m1, nitrogen mustard at 8.0 meg/ml. and S-fluoro uracil at meg/ml.

The following examples are given by way of illustra tion and are not intended to [depart from the spirit and scope of the appended claims.

Example I A nutrient medium having the following composition is prepared and sterilized:

Grams Glucose 1O Soy bean meal 15 Distillers solublcs 2.5 Sodium chloride 2.0 Potassium phosphate dibasic 2.5 Calcium carbonate 2.0

Water to make 1.0 liter.

The medium is inoculated with ATCC 14500 culture and incubated at 28 C. for 65 hours with agitation and aeration. The broth is then filtered.

Example II The experiment of Example I is repeated, substituting a medium of the following composition:

Water to make 1 liter.

1 A commercially available glucose.

2 A commercially available casein hydrolysate.

3 A commercially available concentrated blackstrap m0- lasses stillage from an ethyl alcohol fermentation.

Fermentation is permitted to continue at 30 C. for 72 hours with aeration and agitation. The broth is then filtered.

Example III The filtered broth of Example I is extracted at pH 4 with a half volume of n-butanol. The extract is concentrated azeotropically to remove most of the solvent, thereafter, the concentrate is freeze dried and extracted with an equal volume of methanol. This extract is passed through a column of diethylaminoethyl cellulose and the column is washed with methanol.

In this manner, toyocamycin passes through the column while most of the dark colored impurities remain absorbed on the cellulose. The effiuent fractions are concentrated to a small volume and toyocamycin precipitates as a solid crystalline product which is separated by filtration. Further purification of this product is achieved by recrystallization from a 1:1 mixture of methanol and chloroform.

Example IV The filtered broth of Example 11 is stirred for 1 hour with 2.0% of activated carbon. The activated carbon is rcrnoved by fiitration and is Washed twice with 109 nil. or" a mixture containing three parts of methanol and two parts of an equal mixture of Water and pyridine. Thereafter, the combined extractions are concentrated under reduced pressure and passed t ough a column of activated magncsim sincate. This coismn is Wa hed with 160 m1. of water and the toyocamycin eiuated with 175 m1. of methanol. The nent is concentrated under vacuum and the resulting precipitate recrystaiiized from an equal mixture of methanol and chloroform. in this manner, is obtained pure crystalline toyocaniycin.

Example V Forty-two Swiss Whi e roice Weighing from l8 to 22 grams were impianted Wit L-lZlO by intraperitoneai injection eznpioying the procedure described by Law, L. W., in the Journal of Natl. Cancer Inst, vol. 16, p. 179 (1949). Thereafter, the animals were divided into seven grot containing six an'rnais in each group. Twenty-four hOul'S after the implant tion of L-12l0, six of the animal groups "ere placed toyocarnycin therapy which. was admin istered by intraperitoneai injection while the seventh group of animals Was employed as a control. The details and results of this experiment are listed in Table II.

TABLE 11 Change in Body Weight (gin) Number of Route of Treatments Administration Group Dose,

Control...

The results in Tahie H demonstrated that all the animal groups receiving toyocaniycin therapy had a longer survival time than the control group.

Example VI 6 TABLE In Group Dose,

ingi/lrg.

Number of Treatments Control." 1

Table Hi shows that those animals receiving toyoherapy survived longer the controis.

ces rz'moszrs minre ATCC 14580 in an aqueous nutrient media-n1 conipris n a source of nitrogen, at source of minerals until substantial r ed to the medium.

vation toyoearnyci extraction of said 2 about 4 to about n alkanol at a pH of from n of said extract. on 2 wherein toyocaniyhnnn chromatography on a no resin in ethanol solution. 4. A process as desci ad in ciairn 3 wherein the toyocaroycin eluate from M l coir l is concentrated and oy recrystaiiization from a system cornnol and chloroform. 5. A pro sea as described in ciaim. 1 whei toyocaniycin is pin 12d filtration of said cuitivated broth, treatment of said filtrate with activated carbon, xtraction of in the tile of this patent UNITED STATES PATENTS 2.5263 8 Sooin luiy 18, 1950 2,871,166 Goodman Tan. 27, 1959 

1. A PROCESS FOR THE PREPARATION OF THE TUMOR INHIBITING AGENT, TOYOCAMYCIN, WHICH COMRPISES CULTIVATING STREPTOMYCES RIMUS CULTURE ATCC 14500 IN AN AUEOUS NUTRIENT MEDIUM COMPRISING A SOURCE OF NTIROGEN A SOURCE OF CARBOHYDRATE AND A SOURCE OF MINERALS UNITL SUBSTANTIAL ANTIBIOTIC INHIBITING ACITIVTY IS IMPARTED TO THE MEDIUM. 